The overall aim of this grant proposal is to understand the basis of endothelial dysfunction following myocardial ischemia/reperfusion (I/R) injury. We propose that endothelial dysfunction during myocardial I/R injury is caused by over-expression of tumor necrosis factor-alpha (TNF-alpha). The first aim will casually determine the role of TNF-alpha in endothelial injury following myocardial I/R. We propose that myocardial reperfusion injury to the endothelium will be modest in TNF-alpha knockout (TNF-/-) mice, severe in TNF-alpha over-expression (TNF++/++) mice, and moderate in heterozyqote (TNF-/++, cross of TNF-/- and TNF++/++) mice. We will determine if administration of an antibody to TNF-alpha at the time of reperfusion will prevent endothelial dysfunction as indicated by preservation of endothelial function and maintenance of endothelial barrier function by preventing influx of inflammatory cells. This protocol is designed to mimic a clinically relevant paradigm in which a neutralizing antibody against TNF-alpha could be delivered at the time of a recanalization procedure or restoration of flow following coronary bypass. We will also establish the cell type(s) expressing TNF-alpha during I/R. The second aim will determine the production and sources of reactive oxygen species (ROS) during basal conditions and during I/R injury. This aim will establish the link between TNF-alpha and ROS in the induction of endothelial injury following myocardial I/R. We will determine if administration of an antibody to TNF-alpha at the time of reperfusion will prevent ROS generation. We will establish if the production of ROS is elevated in TNF++/++ mice, or is reduced in TNF mice. We further propose that TNF-alpha will stimulate production of these chemical species by activation of xanthine oxidase and NADPH oxidase. We will also establish the link between TNF-alpha and the activation of superoxide generating enzymes using electron paramagnetic resonance spectroscopy to quantitatively measure O2. production from coronary arterioles during exposure to varying doses of TNF-alpha. The third aim will determine if TNF-alpha expression affects the expression of arginase in endothelial cells. This aim will focus on the proposition that TNF++/++ mice will show elevated arginase expression; whereas, TNF-/- mice will demonstrate reduced levels of arginase expression compared to WT control animals. We utilize a combination of approaches involving in vitro microscopy, fluorescence and EPR analysis of O2. production, electrochemical detection of authentic NO, real time PCR of RNA transcripts, and Western Blotting to evaluate expression of key proteins in the models. We believe that this study will provide a new approach for the treatment of I/R injury and related disorders (post-bypass complications). It is our goal that these studies will provide a clear understanding of the basis for endothelial dysfunction following I/R injury and we hope that this better understanding will facilitate new avenues of therapy to combat this complication.